Many situations exist where qualitative and quantitative detection of the presence of an analyte in a sample is desired. Situations where analyte detection is desirable arise in diverse industries, including: 1) the health care industry, e.g. in clinical and diagnostic medicine; 2) the food processing and chemical industries, e.g. in quality control for food production; and 3) the environmental control industry, e.g. monitoring for the presence of various pollutants in air, ground water or soil.
Many devices and protocols which detect the presence of analytes through chemical and physical means have been developed. Immunoassays make up one broad field of assays which find use in the detection of analytes. In immunoassays, the occurrence of binding events between specific binding pair members is used as an indication of the presence of analyte in the sample. Benefits of using immunoassays in analyte detection include high sensitivity, high specificity, reliability and short assay times.
The binding events which are detected in immunoassays often occur at the surface of a solid support. The time required for a particular immunoassay will depend on the ability of free reactants in the assay medium to reach and bind to the support surface. The ability of the free reactants to reach reactant on the support surface is dependent on many factors including the concentration of reactant on the surface of the support and the surface to volume ratio of the sample support combination. One method of decreasing the time required for an immunoassay is to use a higher concentration of bound reactant on the support surface. Another approach is to increase the ratio of the surface area of the support compared to the volume of sample assayed.
Toward this end, one type of immunoassay which has been demonstrated in capillaries is the ELISA immunoassay. Conventionally, ELISA assays have been conducted in microtiter plates consisting of wells. In ELISA immunoassays, binding events of interest are detected through the appearance of detectable product produced by an enzyme. The formation of a detectable product can be amplified to the extent required by increasing the concentration of the substrate and/or increasing the reaction time. Therefore, there is the opportunity to greatly increase the signal with only a few enzymes becoming bound.
With ELISA immunoassays conducted in capillaries, rapid quantitative results are reported. The reported ELISA assays are described as sensitive and able to detect small amounts of analyte. See Nagianis et al., "A Rapid Quantitative Capillary Tube Enzyme Immunoassay for Human Chorionic Gonadotropin in Urine," Clin. Chem. Acta (1986) 273-279. However, there are disadvantages inherent in ELISA assays. In ELISA immunoassays, a multistep protocol is required using measured reagents. The steps include sample addition, enzyme conjugate addition, incubation, substrate addition, and washing steps. This multiplicity of procedural steps increases the probability of error in the overall assay procedure, particularly when the result may be substrate concentration and/or time sensitive. Also, enzyme reactions tend to be temperature sensitive, which requires temperature control. In addition, the enzyme label is not directly detectable. Instead, one must allow for the detectable product to be produced. Further, ELISA protocols may not be suited for all assays on all types of liquids, where the liquid comprising the analyte of interest may also contain contaminants which interfere with one or more individual steps in the assay, e.g. enzyme activity, detectability of enzyme product, and the like.
Thus, interest exists in the development of assays requiring simple protocols, a minimum of required measurements and small sample volumes, where the assays are relatively insensitive to variations from an optimized protocol, as well as variations in ambient conditions.
Relevant Literature
U.S. Pat. Nos. 5,204,525; 5,164,598; 5,144,139; 5,140,161; 5,004,923; 4,963,498; 4,948,961; 4,756,884 describe combining a sample with reagents, introducing the sample into capillaries, and measuring various agglutination events in the capillaries.
U.S. Pat. Nos. 4,844,869; 4,909,990 and 5,152,962 describe an immunoassay apparatus having an optical fiber inside a hollow tube, where the space between the fiber and tube holds a volume of sample to be assayed. Binding events occur on the surface of the optical fiber and are indicative of the presence of analyte in the sample being assayed. The binding events are detected through changes in total internal reflection of light in the optical fiber.
Other references of interest include: Nagianis et at., Clinica Chimica Acta (1986) 160:273-279; Badley et al., Phil. Trans R. Soc. Lond. (1987) 316: 143-160; Healey et al., Clinica Chimica Acta (1983) 134: 51-58; and Chander & Hurrell, Clinica Chimica Acta (1982) 121:225-230.
Hagamuchi et al., J. Eur. Biochem. (1976) 71: 459-467, describes the preparation of capillary tubes for use in ELISA immunoassays.